The molecular mechanisms of resistance to viral entry fusion inhibitors targeting HIVgp41 are not well-understood. Fundamental gaps in knowledge of the energetic and structural interactions which drive binding hamper the long-term goal of development of new drugs with improved resistance profiles. The overall objective of this application is to (1) develop computational structural models to quantify binding for known gp41 fusion inhibitors (both peptides and small molecules), (2) characterize origins of resistance profiles to current inhibitors, and (3) discover new small molecule drug-leads. Based on strong preliminary results, the central hypothesis is that specific interactions within a conserved hydrophobic pocket on gp41, not exploited by the only currently available anti-fusion drug (peptide inhibitor T20), confer improved resistance profiles to next-generation peptide inhibitors and drive binding for small molecule inhibitors. The rationale for the proposed research is that robust computational models allow drug binding to be fully characterized at the atomic level, and this will enable development of HIV drugs with favorable resistance profiles. Thus, the work proposed is directly relevant to the NIH plan for basic and applied research towards discovery and development of novel agents and therapeutic strategies directed against viral factors involved in HIV replication and persistence. The work employs all-atom computer simulations (molecular dynamics and docking), in conjunction with detailed energetic and structural analysis, to test the central hypothesis and accomplish the goals set forth in each specific aim. Aim #1 will determine the molecular basis of resistance to current peptide fusion inhibitors of gp41 to test the hypothesis that binding affinity for T20 is driven primarily by interactions with mutation-prone regions along the binding interface. Aim #2 will characterize the mechanism of action for reported small molecule inhibitors of gp41 which we postulate are due to specific energetic and structural interactions modulated within the conserved pocket. Aim #3 will identify new small organic molecules, which bind specifically to the gp41 pocket, using virtual-high-throughput-screening in conjunction with experimental validation. Active compounds will be characterized structurally using NMR and X-ray crystallography and developed further. The proposal's contributions are significant because results from detailed binding models and computer simulations will allow the molecular basis of recognition to be delineated, which will enable development of improved fusion inhibitors that maintain activity against clinically relevant HIV escape mutations. PUBLIC HEALTH RELEVANCE: Results from the proposed research will be used to uncover the atomic-level structural and energetic determinates which describe binding of membrane fusion inhibitors with the viral entry protein gp41 which mediates HIV infection. The proposal seeks to understand the origins of resistance to gp41 inhibitors, and develop new compounds with improved resistance profiles, thus the finding are expected to be of direct relevance to public health.